The observed high accuracy in DNA replication and in gene expression cannot be explained on the basis of hydrogen bonding alone. On the level of translation the ribosome plays an active role in a mechanism of proofreading which favors incorporation of cognate transfer RNA over non-cognate transfer RNA. E. coli mutants displaying reduced misreading levels have been isolated during the previous project period. This phenotype has been correlated with increased release factor activity in one of the mutants studied. It remains to be determined whether the mutation involves the structural genes or an inhibitor of release factor activity. The former possibility would permit construction of an overproducer of release factor; the latter possibility identification and purification of an hitherto unknown inhibitor. Other mutants will be further analyzed by in vitro protein synthesis using Q beta RNA as template. A naturally occuring readthrough protein can be detected in this system. This study should reveal potential alterations in efficiency of initiation, elongation rate and readthrough, i.e., misreading at a terminator codon. Mutants with altered ribosomes will be further studied for possible changes in enzymatic binding of cognate and non-cognate transfer RNA. The role of GTP hydrolysis in ribosomal proofreading will be further elucidated using a non-hydrolyzable GTP analogue. It is likely that alteration of the mechanisms responsible for maintaining accuracy of gene expression will have far-reaching consequences for cell physiology. A better understanding of these mechanisms in E. coli will be helpful in evaluating their possible role in eukaryotic cells, particularly with respect to aging and cancer.